Camptothecin compounds and a method of preparation thereof

ABSTRACT

Camptothecin derivatives and pharmaceutically acceptable salts thereof, its manufacturing method and an antineoplastic agent containing it. The camptothecin derivatives are modified in B-ring and E-ring and show improved water solubility and enhanced antineoplastic effect.

TECHNICAL FIELD

This invention relates to camptothecin derivatives and pharmaceuticallyacceptable salts thereof, expressed by the following chemical formula(I), its manufacturing method and antineoplastic agent containing it asan active ingredient

Wherein;

R is hydrogen or —(CH₂)₂—NR₁R₂, (R₁ is hydrogen or a general protectinggroup of amine; R₂ is a lower alkyl, hydroxyethyl or acetoxyethyl;further, R₂ may form heterocyclic compound by binding with an adjacentnitrogen);

n is 1 or 2; R₃ is hydrogen or —OR₄ [R₄ is hydrogen or

(R₅ is methyl or CH₃OCH₂—);

(R₆ is isopropyl, phenyl or —CH₂CH₂Cl.); CH₂OR₇ (R₇ is methyl, ethyl orCH₃OCH₂CH₂—)];

Wherein;

When n is 2 and R₄ is hydrogen, R is not hydrogen;

and when n is 2 and R₃ is H, R is not hydrogen;

also, when R is —CH₂CH₂NHCH₃, R₃ is not hydrogen.

BACKGROUND OF ART

Since the isolation of camptothecin from the Chinese tree, Camptothecaacuminata, by Wall and co-workers (J. Am. Chem. Soc., 1966, 88, 3888),there had been many approaches to synthesize camptothecin. However, thedevelopment of camptothecin as an effective antineoplastic agent wasunsuccessful due to its severe toxicity in the first clinical trial in1970.

Thereafter, Liu et al. reported in 1985 that camptothecin had a specificmode of action to inhibit topoisomerase I. Thus, considerable interesthas focused on this compound. In particular, since topoisomerase Iinhibitor itself was not used clinically, it is expected that with itsactual development, camptothecin may be very efficiently used incombination with other antineoplastic chemotherapeutics having differentmodes of action.

Recently, various studies for the development of camptothecinderivatives have been proposed in order to reduce the toxicity ofcamptothecin and to further enhance its antineoplastic activities. Amongthese related studies, the clinical trial of CPT-11 (Irinotecan)synthesized by Yakurt-Honsha Co. of Japan in 1986 showed that itexhibited excellent antineoplastic activities with less toxicity(Japanese Patent Laid Open Publication No. 64-61482) and followed byother pharmaceutical companies such as Smithkline Beecham (Topotecan)and Glaxo (MDO-camptothecin and 9-aminocamptothecin). Among them, CPT-11is launched. In particular, it is noticeable that said CPT-11 compoundhas exhibited excellent antineoplastic activities in the treatment ofincurable solid tumors such as the lung cancer.

Since a majority of camptothecin derivatives developed hitherto weresemi-synthetic compounds which were obtained from the chemicalmodification of camptothecin, there were some difficulties in procuringcamptothecin and in developing derivatives with a variety of structuresbecause of restrictions of said chemical modification. In addition,these reported total synthesis are not satisfactory in view of bothchemical and optical yields.

DISCLOSURE OF INVENTION

Accordingly, the inventor et al. have confirmed that through the totalsynthesis for a series of novel compounds expressed by said generalformula (I) and followed by in vitro test, these compounds withdifferent mode of action may be used as effective antineoplastic agents.Thus, the present invention has been completed.

Camptothecin is a fused ring system, composed of a quinoline (A and B),fused to a pyrrolidine ring (C), fused to an alpha-pyridone ring (D)which in turn is fused to a lactone ring(E).

In particular, further careful review has been made on camptothecin'smode of action to inhibit topoisomerase I and various structures ofcamptothecin derivatives developed hitherto, and attempts have been alsomade in such a manner to modify the substituents of B-ring or E-ring incamptothecin.

In consideration of the fact that the conventionally developedderivatives have suffered from some side effects such as nausea,vomiting and cystitis due to extremely poor water solubility, theintroduction of side chain at B-ring of camptothecin was made in such amanner that in order to enhance the solubility. Hence, the side chainincluding amino groups was introduced at the 7-position of camptothecin.

In view of the mode of action suggested by Crow that camptothecin bindscovalently with topoisomerase I [J. Med. Chem., 1992, 35, 4160-4164],the modification of camptothecin at E-ring was attempted so as toenhance its reactivity.

The processes for manufacturing camptothecin derivatives and itsintermediates are described in more detail as set forth hereunder, inaccordance with the practice of this invention.

The compounds based upon EXAMPLES can be prepared in accordance with thepractice of this invention as specified in the following Scheme 1.

Wherein;

R, n and R₃ are the same as described in the above and R′ is hydrogen or—(CH₂)₂—NR₁R₂; R₁ is a general protecting group of amine; R₂ is the sameas described in the above. Friedlander condensation of aminoketonecompound (II) with tricyclic Ketone (III) affords the compound expressedby the general formula (I) [organic reactions, 28, 37-202, Wiley & SonsInc, New York (1932)].

The condensation of the compounds (II) and (III) is conducted, ingeneral, in the presence of acid at room temperature or under theheating condition.

The following inert solvents should be used for the reactions so as notto affect the reactions, for example, aromatic hydrocarbons(toluene,benzene, xylene etc.), hydrocarbon halide(dichloromethane, chloroform,1,1-dichloroethane, 1,2-dichoroethane etc.), lower alcohols, amides(N,N-dimethylformamide etc.), or acetic acid.

The reactions may be conducted using inorganic acid(hydrochloric acid orsulfuric acid) or organic acids (methanesulfonic acid,trifluoromethanesulfonic acid, ρ-toluenesulfonic acid, acetic acid,etc.).

The reaction time ranges from 1 to 48 hrs, and the reaction temperatureis 30 to 150° C.

As a typical reaction condition, it is most preferable that reflux ismade available in the presence of ρ-toluenesulfonic acid in toluene.

When R is —CH₂CH₂NHR₂ (R₁ is hydrogen) in the synthesis of generalformula (I), said amino protecting group is removed through thecatalytic hydrogenation using platinum or palladium.

When R′ is hydrogen in the compound (II), compound(I) is obtained aftercarbonyl group of the compound(II) is protected by acetalization withethylene glycol or by formation of shiff base with ρ-toluidine accordingto known process. [Chem. Ber. 76, 1099(1943)].

When R₃ is hydroxy group in the compound (III), thus obtainedcompound(I), wherein R₃ is hydroxy group, is reacted with such reagentsas

and R₇OCH₂Cl to produce the compound(I) wherein the hydroxy group of R₃is thus modified.

In particular, when R₆NCO is used, the compound expressed by the generalformula (I) can be obtained by using the catalytic amounts of tincomplex (e.g., di-n-butyltin diacetate, etc.).

The aminoketone (II) can be prepared in accordance with Scheme 2.

Wherein;

R′, R₁ and R₂ are the same as described in the above.

A novel aminoketone compound (II) of this invention can be prepared asfollows:

2′-nitroacetophenone reacts with monoalkylamine (e.g., ethylamine,propylamine or isopropylamine), monoarylamine (e.g., benzylamine) ordialkylamine (e.g., morpholine, piperidine or diethylamine) in thepresence of conc. hydrochloric acid, through Mannich reaction withparaformaldehyde to give the compound(IV).

The reaction is conducted at 30-80C for 1 to 48 hrs.

In case of the compound (IV) having monoalkylamine or monoarylamine, thegeneral protecting group of amine is introduced to give the saidcompound (IV).

According to this invention, Cbz (carbobenzyloxy) is a preferableprotecting group of amine.

The compound (IV) is treated with sodium dithionite in lower alcoholsolvent to give the compound (II).

The reaction is conducted at 30 to 100° C. for 1 to 10 hrs.

As specified in Scheme 3 below, a novel tricyclic ketone (III) of thisinvention can be prepared from the compound (IX) which is already known.

From the compound (III), tricyclicketone (n is 2 and R₃ is H) is alreadyknown compound prepared in accordance with a convention method (U.S.Pat. No. 4,894,456 (1990)).

Wherein;

R₃T n, R₅ and R₆ are the same as described in the above and R₈ is thegeneral protecting group of hydroxyl group.

During α-alkylation re action where n is 2, the compound (IX) reactswith base such as KOtBu or NaH in inert solvent including N,N-dimethylformamid or 1,2-dimethoxyethane. Then, the reaction mixture isreacted with 2-bromoethanol protected with tetrahydropyranyl,methoxymethyl or methoxyethoxymethyl to give the compound (VIII).

The reaction is preferably conducted at 30 to 60° C. for 18 to 48 hrs.

Meanwhile, when n is 1, α-hydroxymethylation is conducted usingformaldehyde in alcoholic solvent and followed by the protection withthe general protecting group of OH to give the compound(VIII).

The preferable protecting group of this invention is methoxymethyl ormethoxyethoxymethyl.

The compound (VII) was obtained by the reduction of compound (VIII)using Raney-Nickel as a catalyst in a cosolvent of acetic acid andacetic anhydride.

The reaction is conducted at 30 to 80° C. for 1 to 10 hrs.

The rearrangement via nitroso compound is done by reacting the compound(VII) with sodium nitrite in a co-solvent of acetic anhydride and aceticacid at 0 to 50° C. for 1 to 24 hrs. Thus, obtained nitroso compound isrefluxed in the solvent such as CCl4 at 60 to 90° C. for 5 to 18 hrs togive the compound (VI).

When n is 1 or 2, the reaction route of the diester (VI) becomesslightly different. In case that n is 2, compound(VI) is hydrolyzed inalkali aqueous solution including LiOH, NaOH and KOH in methanol andtreated with acidic aqueous solution such as acetic acid or hydrochloricacid to give the compound (Vc).

Said hydrolysis is conducted at 0 to 50° C. for 30 mins to 5 hrs.

The lactonization in acidic aqueous solution is conducted at 0 to 50° C.for 1 to 48 hrs.

Compound (Vc) is treated with base such as potassium t-butoxide in theamine solvents such as N,N-dimethylformamide or N,N-dimethylacetamideand oxidized with the bubbling of O₂ under triethylphosphite to give thecompound (V).

The reaction is conducted at −10 to 50° C. for 30 mins to 5 hrs.

Meanwhile, when n is 1, compound(VI) is treated with organic base suchas DBU, DBN or triethylamine in aromatic hydrocarbon solvent such asbenzene, toluene or xylene to produce the compound (Va).

The reaction is conducted at 0° C. to 100° C. for 10 mins to 5 hrs.

The compound (Va) is treated with OsO₄ in a pyridine solvent to give thecompound (Vb). This reaction is conducted at 10 to 50° C. for 1 to 10hrs. The lactonization of the compound (Vb) is carried out as the sameas described in the above.

The compound (V) where n is 2, is treated with acid to produce compound(III). The reaction is conducted at 10 to 80° C. for 30 mins to 10 hrs.In case of the compound (V) wherein n is 1, said deketalization is doneafter introducing the substituent by treating

for the preparation of the compound (III).

In case of the compound (I) of this invention where R is CH₂CH₂NR₁R₂,the conversion of secondary amine into the salts of inorganic acids(e.g., hydrochloric acid, sulfuric acid, phosphoric acid, etc.) ororganic acids (e.g., acetic acid) makes it possible to preparephysiologically acceptable salts.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 illustrates the difference of ILS(%) in various dose between thecompound prepared in example 30 and camptothecin.

FIG. 2 illustrates the inhibitory effects on mice melanorma cells inaccordance with the compound prepared in example 30.

BEST MODE FOR CARRYING OUT THE INVENTION

This invention is explained in more detail by the following examples andexperiments, but the claims are not limited to these examples andexperiments.

EXAMPLES 1

Preparation of 3-morpholino-1-(2′-nitrophenyl)propan-1-one

A solution of morpholine (2.27 g, 0.026 mol) in ethanol (10 ml) wastreated with c-HCl (3 ml) for 10 mins, concentrated under reducedpressure to prepare morpholine HCl salt. To this mixture,2′-nitroacetophenone (3.3 g, 0.02 mol), paraformaldehyde (0.8 g, 0.029mol), c-HCl (0.1 ml) and anhydrous ethanol (10 ml) were added. Thereaction mixture was stirred under reflux for 30 hrs. This reactionmixture was then cooled to room temperature, alkalized with 10% sodiumcarbonate solution, and the reaction mixture was extracted withdichloromethane(3×50 ml). The organic layer was dried over anhydroussodium sulfate and concentrated under reduced pressure to give the crudeproduct. The crude product was purified by flash column chromatographywith 5% ethanol in dichloromethane to give the desired product (1.5 g,35%) as an oil.

¹H-NMR (CDCl₃) δ 3.1 (m, 3H), 3.5 (m, 4H), 3.75 (m, 3H), 3.99 (m, 2H),7.85 (m, 3H), 8.15 (d, 1H, J=8 Hz)

EXAMPLE 2

Preparation of 3-piperidino-1-(2′-nitrophenyl) propan-1-one

In the same procedure as in EXAMPLE 1, the desired product (458 mg, 29%)was yielded using piperidine (515 mg, 6.06 mmol), 2′-nitroacetophenone(1 g, 6.06 mmol), paraformaldehyde (260 mg, 8.67 mmol); c-HCl (1 ml) andanhydrous ethanol.

¹H-NMR (CDCl₃) δ 1.5 (m, 6H), 2.38 (t, 4H, J=5 Hz), 2.76 (t, 2H, J=6Hz), 3.00 (t, 2H, J=6 Hz), 3.99 (m, 2H), 7.40-7.75 (m, 3H), 8.13 (d, 1H,J=8 Hz)

EXAMPLE 3

Preparation of 3-pyrrolidino-1-(2′-nitrophenyl) propane-1-one

In the same procedure as in EXAMPLE 1, the desired product (347 mg, 23%)was yielded using pyrrolidine (560 mg, 7.88 mmol), 2′-nitroacetophenone(1 g, 6.06 mmol), paraformaldehyde (260 mg, 8.67 mmol), c-HCl (1 ml) andanhydrous ethanol (5 ml).

EXAMPLE 4

Preparation of3-(N-benzyl-N-carbobenzyloxyamino)-1-(2′-nitrophenyl)propane-1-one

A solution of benzylamine (2.79 g, 0.026 mol) in ethanol (10 ml) wastreated with c-HCl (3 ml) for 10 min, concentrated under reducedpressure to prepare benzylamine·HCl salt.

To this solution, 2′-nitroacetophenone (3.3 g, 0.02 mol),paraformaldehyde (800 mg, 0.029 mol), c-HCl (0.1 ml) and anhydrousethanol (10 ml) were added.

The reaction mixture was stirred under reflux for 30 hrs. This reactionmixture was then cooled to room temperature, alkalized with 10% sodiumcarbonate solution, and the reaction mixture was extracted withdichloromethane (3×50 ml) The organic layer was dried over anhydrousmagnesium sulfate and concentrated under reduced pressure to give thecrude product, which was dissolved in dichloromethane and cooled to 0°C. To this mixture, triethylamine (1.2 g, 0.012 mol) was added andfollowed by another dropwise addition of benzylchloroformate (2 ml). Thereaction mixture was stirred under nitrogen flow at 0° C. for 1 hr,heated to room temperature and then stirred for additional 5 hrs.

After removing the solvent under reduced pressure, the mixture wasdissolved in ethylacetate (100 ml). The organic layer was washed withwater (3×30 ml) and brine (30 ml) and dried over anhydrous sodiumsulfate. After the filtration, the crude product was obtained byremoving the solvent. The residue was purified by flash columnchromatography with n-hexane-ethylacetate(1:10) to give the desiredproduct (1.5 g, 36%).

¹H-NMR (CDCl₃) δ 3.0 (m, 2H), 3.70 (t, 2H, J=7 Hz), 4.60 (s, 2H), 5.17(s, 2H), 7.10-7.70 (m, 13H), 8.10 (d, 1H, J=8 Hz)

In the same procedure as in EXAMPLE 4, the compounds of EXAMPLE 5-8 wereprepared and the results were as follows:

EXAMPLE R ¹H-NMR:δ 5 CH₂CH₃ 8.102(d, 1H, J=1.2Hz), 7.596- 7.315(m, 8H),5.125(s, 2H), 3.762-3.61(m, 4H), 1.853(t, 2H, J=6.4Hz), 1.159(t, 3H,J=7.2Hz) 6 CH₂CH₂CH₃ 8.124(d, 1H, J=1.2Hz), 7.700- 7.272(m, 8H),5.125(s, 2H), 3.717(t, 2H, J=3.6Hz), 3.322- 3.279(m, 2H), 1.624-1.546(m,4H) 0.894(t, 3H) 7 CH(CH₃)₂ 8.115(d, 1H), 7.613-7.261(m, 8H) 5.135(s,2H), 4.488-4.210(m, 1H) 3.639(t, 2H, J=7.2Hz), 1.260(t, 2H, J=7.2Hz),1.165(d, 6H, J=6.4Hz) 8 CH₂CH₂OH 8.119(d, 1H, J=8.0Hz), 7.623- 7.261(m,8H), 5.127(s, 2H), 3.802-3.719(m, 2H), 3.557(t, 2H, J=5.2Hz),3.392-3.353(m, 2H), 3.068-3.051(m, 2H)

EXAMPLE 9

Preparation of3-(N-2′-acetoxyethyl-N-carbobenzyloxyamino)-1-(2′-nitrophenyl)propane-1-one

A mixture of 3-[(N-(2′-hydoxyethyl)-N-carbobenzyloxyamino)]-1-(2′-nitrophenyl)propane -1-one (40 mg, 0.107 mmol) and4-dimethylamino pyridine (1.2 mg, 0.01 mmol) was dissolved in anhydrousdichloromethane(2 ml), then added triethylamine (0.02 ml, 0.160 mmol)and acetic anhydride(0.02 ml, 0.214 mmol). The reaction mixture wasstirred at room temperature for 30 mins and washed with a saturatedammonium chloride aqueous solution, water and brine successively. Then,the organic layer was dried over anhydrous magnesium sulfate andconcentrated under reduced pressure. The residue, so obtained, waspurified by column chromatography with ethylacetate-n-hexane (1:3) togive the desired product (40 mg, 90%) as a yellow oil.

¹H-NMR (400 MHz, CDCl₃) δ 8.123 (d, 1H, J=8.4 Hz), 7.607-7.261 (m, 8H),5.136 (s, 2H), 4.194 (m, H), 3.784-3.754 (m, 2H), 3.660-3.646 (m, 2H),3.172 (m; 2H), 2.047 (s, 3H)

EXAMPLE 10

Preparation of 3-morpholino-1-(2′-aminophenyl)propane-1-one

To a solution of 3-morpholino-1-(2′-nitrophenyl) propane-1-one(200 mg,0.758 mmol) dissolved in 10 ml of 95% ethanol was added sodiumdithionite(660 mg, 3.79 mmol) and the reaction mixture was stirred underreflux for 3 hrs. The reaction mixture was concentrated under reducedpressure. The residue was purified by flash column chromatography withethanol-dichloromethane-triethylamine to yield the desired product (174mg, 89%) as an oil.

¹H-NMR (CDCl₃) δ 3.20 (m, 3H), 3.50 (m, 3H), 3.5 (m, 4H), 3.99 (m, 2H),7.5-7.8 (m, 3H), 8.08 (d, 1H, J=8 Hz)

EXAMPLE 11

Preparation of 3-piperidino-1-(2′-aminophenyl) propane-1-one

The same procedure as in EXAMPLE 10 was applied to the compound ofEXAMPLE 2 (200 mg, 0.758 mmol) and sodium dithionite(660 mg, 3.79 mmol)so that the desired product (174 mg, 89%) was yielded.

EXAMPLE 12 Preparation of 3-pyrrolidino-1-(2′-aminophenyl)propane-1-one

The same procedure as in EXAMPLE 10 was applied to the compound ofEXAMPLE 3 (400 mg) and sodium dithionite (1.052 g, 6.05 mmol) so thatthe desired product (235 mg) was yielded.

In the same procedure as used in EXAMPLE 10, the compounds of EXAMPLE13-17 were prepared and the results were as follows:

EXAM- PLE R₂ R₁ ¹H-NMR:δ 13 CH₂CH₃ Cbz 7.365-7.262(m, 9H), 5.144(s, 2H),3.764-3.71(m, 4H), 1.871-1.838(m, 2H), 1,253(t, 3H, J=7.2Hz) 14CH₂CH₂CH₃ Cbz 7.885-7.230(m, 9H), 5.142(s, 2H), 3.668-3.613(m, 2H),3.265(t, 2H, J=6.4Hz), 1.616-1.513(m, 4H), 0.895(t, 3H) 15 CH(CH₃)₂ Cbz7.496(d, 1H, J=7.28Hz), 7.377- 7.349(m, 3H), 7.299-2.227(m, 5H) 5.172(s,2H), 4.376-4.216(m, 1H), 3.534-3.371(m, 4H), 1.240-1.044(m, 6H) 16 CH₂PhCbz 3.17(m, 2H), 3.65(m, 2H), 4.55(s, 2H), 5.10(s, 2H), 6.20(brs, NH₂),7.10-7.70(m, 13H), 8.10(d, 1H, J=8Hz) 17 CH₂CH₂OAc Cbz 7.771(d, 1H,J=7.6Hz), 7.574- 7.262(m, 8H), 6.641(d, 2H, J=7.6 Hz), 5.114(s, 2H),4.186(m, 2H) 3.773-3.169(m, 6H), 2.042(s, 3H)

EXAMPLE 18

Preparation ofdl-7-[2-(N-benzyl-N-carbobenzyloxyamino)ethyl]camptothecin

A mixture of 3-(N-benzyl-N-carbobenzyloxyamino)-1-(2′-aminophenyl)propane-1-one(543 mg, 1.4 mmol) and4-ethyl-6-oxo-1,4,7,8-tetrahydro-4-hydroxy-pyrano[3,4-f]indolizine-3,10(6H)-dione (263 mg, 1.0 mmol) was dissolved intoluene (50 ml). The reaction mixture was stirred under reflux for 30mins, and ρ-toluenesulfonic acid (25 mg, 0.13 mmol) was added to themixture. The reaction mixture was stirred in a Dean-Stark trap underreflux for 9 hrs. The solvent was removed under reduced pressure and theresidue was purified by flash column chromatography withethylacetate-dichloromethane (1:1) to give the desired product (507 mg,59%) as a yellow powder.

¹H-NMR (CDCl₃) δ 1.04 (t, 3H, J=8 Hz), 1.90 (m, 2H), 3.10-3.90 (m, 4H),4.53 (s, ½×2H), 4.61 (s, ½×2H), 4.95 (s, ½×2H), 5.07 (s, ½×2H), 5.30 (s,2H), 5.31 (d, 1H, J=16 Hz), 5.75 (d, 1H, J=16 Hz), 7.20-8.20 (m, 15H)

In the same procedure as in EXAMPLE 18, the compounds of EXAMPLE 19 to24 were prepared and the results were as follows:

EXAM- PLE R₂ R₁ ¹H-NMR(CDCl₃)δ 19 CH₂CH₂CH₃ Cbz 8.236-7.261(m, 10H),5.757, 5.324 (ABq, 2H, J=16Hz), 5.293(s, 2H), 5.193(s, 2H),3.593-3.199(m, 4H) 1.942-1.863(m, 2H), 1.634-1.516 (m, 4H), 1.047(t, 3H,J=7.2Hz), 0.944-0.842(m, 3H) 20 CH(CH₃)₂ Cbz 8.197(d, 1H),7.926-7.628(m, 3H) 7.485-7.365(m, 6H), 5.694(d, 1H, J=13.08Hz),5.552-5.270(m, 3H) 5.246(s, 2H), 4.476-4.310(m, 1H), 3.514-3.456(m, 4H),1.997-1.910 (m, 2H), 1.248-1,208(m, 6H), 1.044(t, 3H, J=5.92Hz) 21CH₂CH₂OAc Cbz 8.344-7.261(m, 10H), 5.748(d, 1H J=16.8Hz), 5.308(d, 1H,J=16.8Hz) 5.298(s, 2H), 5.113(s, 2H), 4.255-4.126(m, 2H), 3.791-3.371(m, 6H), 2.041(s, 3H), 1.954- 1.610(m, 2H), 1.042(t, 3H, J= 7.2Hz)

EXAMPLE R₁ R₂ ¹H-NMR(CDCl)δ 22 piperidino ¹H-NMR(200MHz, DMSO-d₆)0.92(t, J=6Hz, 3H), 1.32(m, 6H), 1.90(m, 2H), 2.34(s, 3H), 2.90(m, 2H),3.30 (m, 6H), 5.48(s, 2H), 5.49(s, 2H), 6.58(s, 1H), 7.16(d, J=8Hz, 2H),7.52(d, J=8Hz, 2H), 7.77-8.30(m, 4H) 23 pyrrolidino ¹H-NMR(200MHz,DMSO-d6)0.94(t, J= 6Hz, 3H), 1.25(m, 4H), 1.87(m, 2H), 2.33(s, 3H),2.90(m, 2H), 3.11(m, 6H), 3.53(s, 1H), 5.52(s, 2H), 5.53(s, 2H), 6.60(s,1H), 7.15(d, J=8Hz, 2H), 7.51(d, J=8Hz, 2H), 7.77-8.30(m, 4H) 24morpholino ¹H-NMR(200MHz, CDCl₃)δ0.86(t, J= 6Hz, 3H), 1.89(m, 2H),2.25(s, 3H) 2.85(m, 2H), 3.05(m, 2H), 3.51(m, 4H), 3.71(m, 4H), 5.39(s,2H), 5.40 (s, 2H), 6.49(s, 1H), 7.08(d, J=8Hz, 2H), 7.44(d, J=8Hz, 2H),7.70-8.30(m, 4H)

EXAMPLE 25

Preparation ofdl-7-[2-(N-hydroxyethyl-N-carbobenzyloxyamino)ethyl]camptothecin

A mixture ofdl-7-[2-(N-acetoxyethyl-N-carbobenzyloxyamino)ethyl]camptothecin (28.2mg, 0.05 mmol) and potassium carbonate (25 mg, 0.15 mmol) was dissolvedin mixed a solution of methanol and water (1:1, 1.5 ml) and the reactionmixture solution was stirred at room temperature for 3 hrs. The reactionmixture was acidified with 1N HCl solution, extracted withdichloromethane and washed with brine. The organic layer was dried overanhydrous sodium sulfate, filtered and concentrated under reducedpressure. The residue was purified by flash column chromatography withdichloromethane-methanol (20:1) to give the desired product (17 mg, 60%)as a yellow solid.

¹H-NMR (400 MHz, CDCl₃) δ 8.171-7.762 (m, 10H), 5.710 (d, 2H, J=16.8Hz), 5.308 (d, 2H, J=16.8 Hz), 5.301 (s, 2H), 5.207 (s, 2H), 3.868-3.466(m, 8H), 1.926-1.832 (m, 2H), 1.033 (t, 3H, J=7.6 Hz)

EXAMPLE 26 Preparation of dl-7-[2-(N-benzylamino) ethyl]camptothecin

dl-7-[2-(N-Benzyl-N-carbobenzyloxyamino)ethyl]camptothecin (50 mg, 0.08mmol) was dissolved in glacial acetic acid (20 ml) and treated with 10%Pd/C (25 mg). The reaction mixture was stirred under hydrogen flow of 40psi for 9 hrs, and the catalyst was removed through cellite pad byfiltration under reduced pressure. The filtrate was concentrated underreduced pressure. The residue was purified by flash columnchromatography with 10% methanol-dichloromethane to give the desiredproduct (24 mg, 63%) as a yellow powder.

¹H-NMR (400 MHz, CDCl₃) δ 0.89 (t, 3H, J=8 Hz), 1.87 (m, 2H), 2.90 (m,2H), 3.5 (m, 4H), 3.73 (s, 1H), 5.33 (s, 2H), 5.43 (s, 2H), 6.52 (s,1H), 7.25 (m, 6H), 7.70-8.30 (m, 4H)

EXAMPLE 27

Preparation of dl-7-[2-(N-acetoxyethylamino) ethyl]camptothecin

The same procedure as in EXAMPLE 26, was carried out.

¹H-NMR (400 MHz, CDCl₃+MeOH-d₄) δ 8.323 (d, 1H, J=8.8 Hz), 8.138 (d, 1H,J=8.4 Hz), 7.812 (t, 1H, J=6.8 Hz), 7.675 (t, 1H, J=6.8 Hz), 7.647 (s,1H), 5.742 (d, 1H J=16.4 Hz), 5.324-5.284 (m, 2H), 4.169-4.122 (m, 2H),3.373 (t, 2H, J=7.6 Hz), 3.108-3.040 (m, 4H), 2.090 (s, 3H), 2.024 (s,3H), 1.955-1.803 (m, 2H), 1.033 (t, 3H, J=7.6 Hz)

EXAMPLE 28

Preparation of dl-7-[2-(N-hydroxyethyl amino)ethyl]camptothecin

The same procedure as in EXAMPLE 26, was carried out.

¹H-NMR (CDCl₃+MeOH-d₄) 8.284 (d, 1H, J=8.0 Hz), 8.226 (d, 1H, J=8.8 Hz),7.863 (t, 1H, J=6.8 Hz), 7.756 (t, 1H, J=6.8 Hz), 7.730 (s, 1H), 5.682(d, 1H,J=13.6 Hz), 5.641 (d, 1H, J=13.6 Hz), 5.130 (s, 2H), 3.800-3.190(m, 8H), 1.962-1.898 (m, 2H), 1.031 (t, 3H, J=4.4 Hz), 2.035 (s, 3H)

EXAMPLE 29

Preparation of dl-7-[2-(N-ethylamino)ethyl]camptothecin

A mixture of 2′-amino-2-(N-carbobenzyloxy-N-ethylamino)propiophenone (34mg, 0.11 mmol) and 4-ethyl-6-oxo-1,4,7,8-tetrahydro-4-hydroxy-pyrano[3,4-f]indolizine-3,10(6H)-dione(34 mg, 0.1 mmol) was dissolved in tolueneand was refluxed for 30 mins. After addition of ρ-toluenesulfonic acid(19.2 mg, 0.11 mmol), the reaction mixture was then refluxed in aDean-Stark trap for 3 hrs. The reaction mixture was concentrated underreduced pressure, and the residue was purified by column chromatographywith dichloromethane-methanol (10:1) to give the desired product(40 mg,67%) as a solid.

¹H-NMR (400 MHz, DMSO-d₆) δ 8.280 (d, 1H, J=8.6 Hz), 8.184 (d, 1H, J=7.4Hz), 7.873-7.753 (m, 2H), 7.471 (d, 2H, J=7.2 Hz), 7.111 (d, 2H, J=7.2Hz), 7.356 (s, 1H), 6.523 (s, 1H), 5.439 (s, 2H), 3.534-2.753 (m, 6H),2.217 (s, 3H), 1.671-1.643 (m, 2H), 1.230 (t, 3H, J=6.8 Hz), 0.743 (t,3H,J=7.2 Hz)

EXAMPLE 30

Preparation of dl-7-[2-(N-isopropylamino)ethyl]camptothecin

The same procedure as in EXAMPLE 29 was carried out.

¹H-NMR (400 MHz, DMSO-d₆) δ 8.870-8.567 (m, 2H), 8.298-8.155 (m, 2H),7.867 (d, 2H, J=7.6 Hz), 7.741 (s, 1H), 7.500 (d, 2H, J=7.6 Hz), 5.828(s, 2H), 5.795 (s, 2H), 3.958-3.820 (m, 2H), 2.666 (s, 3H), 2.320-2.200(in, 2H), 1.614 (d, 6H, J=6.4 Hz), 1.533 (t, 2H), 1.267 (t, 3H)

EXAMPLE 31

dl-7-[2-(Morpholino)ethyl]camptothecin trifluoro-acetate

The same procedure as in EXAMPLE 24 was carried out except thattrifluoroacetic acid was used instead of p-toluenesulfonic acid.

¹H-NMR (200 MHz, DMSO-d₆) δ 0.92 (t, J=6 Hz, 3H), 1.94 (m, 2H), 2.90 (m,4H), 3.30-3.65 (m, 8H), 5.47 (s, 2H), 5.48 (s, 2H), 6.56 (s, 1H),7.70-8.30 (m, 4H)

EXAMPLE 32

Preparation of 6-cyano-1,1-(ethylenedioxy)-7-[1′-(ethoxycarbonyl)-3′-(methoxymethyloxypropyl]-5-oxo-Δ⁶⁽⁸⁾-tetrahydroindolizine

6-Cyano-1,1-(ethylenedioxy)-7-[(ethoxycarbonyl)methyl]-5-oxo-Δ⁶⁽⁸⁾-tetrahydroindolizine(608.6 mg, 2 mmol) was dissolved in anhydrous dimethylformamide (3 ml),cooled to 5° C. and after addition of potassium t-butoxide (258.1 mg,2.3 mmol), the reaction mixture was stirred at the same temperature for30 mins. To this mixture, 2-brmoethanolmethoxy-methylether (1.352 g, 8mmol) was added and the reaction mixture was stirred at 45-50° C. for 20hrs. The reaction mixture was diluted with water(50 ml), extracted withdichloromethane (100 ml). The organic layer was washed with water(80 ml)and brine(80 ml), dried over anhydrous magnesium sulfate andconcentrated under reduced pressure. The residue was purified by columnchromatography with ethylacetate-n-hexane (2:1) to give the desiredproduct (734.8 mg, 95%) as a pale yellow solid.

IR(neat) 2224, 1734, 1656 cm⁻¹

¹H-NMR (400 MHz, CDCl₃) δ 1.254 (t, 3H, J=6.8 Hz), 1.987-2.020 (m, 1H),2.410 (t, 2H, J=6.8 Hz), 2.43-2.449 (m, 1H), 3.447-3.485 (m, 1H)3.564-3.603 (m, 1H), 4.117-4.201 (m, 7H), 4.225 (q, 2H J=7.6 Hz),4.584,4.609 (ABq, 2H, J=6.8 Hz),6.367 (s, 1H)

MS(EI) m/e 392 (M⁺), 377 (M⁺−CH₃), 361 (M⁺−OCH₃), 347 (M⁺−C₂H₅O)

EXAMPLE 33

Preparation of6-(acetamidomethyl)-1,1-(ethylenedioxy)-7-[1′-(ethoxycarbonyl)-3°C.-(methoxymethyl oxypropyl)]-5-oxo-Δ⁶⁽⁸⁾-tetrahydroindolizine

250 Drops of Raney-Nickel (50% aqueous slurry) was washed with water (5ml) and acetic acid (10 ml) 5 times respectively and added into a parrbottle.

To this solution, a compound(743.8 mg, 1.9 mmol), obtained in EXAMPLE32, acetic anhydride(30 ml) and acetic acid (10 ml) were added and thereaction mixture was hydrogenated at a pressure of 45 psi and 45-50° C.for 3 hrs. The catalyst was removed by filtration and the filtrate wasconcentrated under reduced pressure. The residue was purified by columnchromatography with dichloromethane-methanol (20:1) to give the desiredproduct(789.5 mg, 95%) as a yellow oil.

IR(neat) 1733, 1660, 1635 cm⁻¹

¹H-NMR (400 MHz, CDCl₃) δ 1.214 (t, 3H, J=6.8 Hz), 1.924 (s, 3H),1.939-1.98 (m, 1H), 2.382 (t, 2H, J=6.8 Hz), 2.399-2.40 (m, 1H), 3.343(s, 3H), 3.44-3.58 (m, 2H), 4.075-4.131 (m, 7H), 4.145 (q, 2H), 4.34,4.39 (d, d, 2H), 4.571, 4.592 (ABq, 2H, J=6.8 Hz), 6.306 (s, 1H), 6.61(brs, 1H)

MS(EI) m/e 438 (M+), 423 (M⁺−CH₃), 407 (M⁺−OCH₃), 395 (M⁺−COCH₃), 393(M′—C₂H₅O)

EXAMPLE 34

Preparation of6-(acetoxymethyl)-1,1-(ethylenedioxy)-7-[1′-(ethoxycarbonyl)-3′-(methoxymethyloxypropyl)]-5-oxo-Δ⁶⁽⁸⁾-tetrahydroindolizine

To a compound (789.5 mg, 1.8 mmol) obtained in EXAMPLE 33, aceticanhydride(15 ml), acetic acid(5 ml) and sodium nitrite(621.2 mg, 9 mmol)were added and the reaction mixture was stirred at 5 to 10° C. for 6hrs. After removing the inorganic material, so formed by filtration, thefiltrate was concentrated under reduced pressure to give nitrosocompound. With addition of carbon tetrachlonride (50 ml), the mixturewas heated under reflux for 18 hrs. The reaction mixture was washed withwater (10 ml) and brine (10 ml), dried over anhydrous magnesium sulfateand concentrated under reduced pressure. The residue was purified bycolumn chromatography. with dichloromethane -methanol(20:1) to give thedesired product (720 mg, 91%) as a colorless oil.

IR(neat) 1740, 1733, 1654 cm⁻¹

¹H-NMR (400MHZ, CDCl₃) δ 1.217 (t, 3H, J=6.8 Hz), 1.86-1.95 (m, 1H),2.056 (s, 3H), 2.368 (t, 2H), 2.38-2.43 (m, 1H), 3.333 (s, 3H),3.41-3.57 (m, 2H), 4.087-4.119 (m, 9H), 4.569 (s, 2H), 5.259 (s, 2H),6.304 (s, 1H)

MS(EI) m/e 439 (M⁺), 408 (M⁺−OCH₃), 396 (M⁺−COCH₃)

EXAMPLE 35

Preparation of1,1-(ethylenedioxy)-(5′-methoxymethyloxyethyl-2′H,5′H,6′H-6-oxopyrano)[3′,4′-f]-5-oxo-Δ⁶⁽⁸⁾-tetrahydroindolizine

A mixture of the compound (720 mg, 1.64 mmol) obtained in prepared byEXAMPLE 34 and methanol (15 ml) was added to LiOH.H₂O (172 mg, 4.1 mmol)dissolved in water (5 ml) and stirred at 25 to 30° C. for 2 hrs. Thereaction mixture was concentrated under reduced pressure to removemethanol and with addition of water(15 ml), dichloromethane(50 ml) andacetic acid (5 ml), the reaction mixture was stirred at 25 to 30° C. for10 hrs. The organic layer was separated from the reaction mixture,washed with water (10 ml) and brine (10 ml) and dried over anhydrousmagnesium sulfate. The organic layer was concentrated under reducedpressure. The residue was purified by column chromatography withdichloromethanemethanol (30:1) to give the desired product (720 mg, 91%)as a white solid.

IR(neat) 1747, 1662 cm⁻¹

₁H-NMR (400 MHz, CDCl₃) δ 2.178-2.216 (m, 2H), 2.404 (t, 2H, J=6.8 Hz),3.371 (s, 3H), 3.599-3.643 (m, 2H), 3.701 (t, 1H, J=6.8 Hz), 4.109-4.192(m, 6H), 4.618 (s, 2H), 5.278, 5.43 (ABq, 2H, J=16.4 Hz), 6.169 (s, 1H)

MS(EI) m/e 351 (M⁺), 320 (M⁺−OCH₃), 307 (M⁺−CO₂)

EXAMPLE 36

Preparation of1,1-(ethylenedioxy)-(5′-methoxymethyloxyethyl-5′-hydroxy-2′H,5′H,6′H-6-oxopyrano)[3′,4′-f]-5-oxo-Δ⁶⁽⁸⁾-tetrahydroindolizine

Potassium t-butoxide (273 mg, 2.43 mmol) was added to a mixture of thecompound (570.3 mg, 1.63 mmol) obtained in EXAMPLE 35 dissolved inanhydrous DMF (20 ml) and stirred at 0 to 5° C. for 30 mins. Thereaction mixture was cooled to −10 to −5° C. and with addition oftriethyl phosphite (974 μl, 5.68 mmol), the mixture was stirred for 2.5hrs, while bubbling the oxygen. Water (10 ml) was added to the mixtureand pH was adjusted to 3.5 with 1N-HCl. Then, the reaction mixture wasextracted with dichloromethane (30 ml×2 times), washed with water (20ml), and brine (20 ml) and dried over anhydrous magnesium sulfate. Theorganic layer was concentrated under reduced pressure. The residue waspurified by column chromatography with dichloromethane-methanol (25:1)to give the desired product (416.4 mg, 69.8%) as a white solid.

IR(neat) 3503, 1749, 1654 cm⁻¹

¹H-NMR (400 MHz, CDCl₃) δ 1.919-1.974 (m, 1H), 2.010-2.063 (m, 1H),2.348 (t, 2H, J=6.8 Hz), 3.30 (s, 3H), 3.542-3.597 (m, 1H), 3.639-3.684(m, 1H), 4.002-4.1541 (m, 7H), 4.516, 4.494 (ABq, 2H, J=6.4 Hz), 5.111,5.557 (ABq, 2H, J=16 Hz), 6.541 (s, 1H)

MS(EI) m/e 367 (M⁺), 336 (M⁺−OCH₃), 323 (M⁺−CO₂)

EXAMPLE 37 :

Preparation of1,5-dioxo-(5′-hydroxyethyl-5′-hydroxy-2′H,5′H,6/H-6-oxopyrano)[3′,4′-f]-Δ⁶⁽⁸⁾-tetrahydroindolizine

A mixture of THF (200 ml), water (1 ml) and 6N HCl (2.5 ml) was added tothe compound (146.1 mg, 0.4 mmol) obtained in EXAMPLE 36 and stirred at50-55° C. for 1 hr. The reaction mixture was concentrated under reducedpressure. The residue was purified by Dianion^(R) HP-20 (Mitsubishi)with acetonitrile-water (4:1) and concentrated under reduced pressure togive the desired product (93.2 mg, 84%) as a black resin.

IR(KBr) 3470, 1749, 1654 cm⁻¹

¹H-NMR (400 MHz, CDCl₃) δ 1.92-2.11 (m, 2H), 2.962 (t, 2H, J=6.0 Hz),3.674-3.86 (m, 2H), 4.326 (t, 2H, J=6.0 Hz), 5.259, 5.627 (ABq, 2H,J=16.8 Hz), 5.287 (s, 1H), 7.284 (s, 1H)

MS(EI) m/e 279 (M⁺), 261 (M⁺−H₂O), 235 (M⁺−C₂H₅OH)

EXAMPLE 38

Preparation of dl-18-hydroxycamptothecin

A solution of 2-aminobenzaldehyde ethyleneacetal (75.6 mg, 0.46 mmol)and toluene (8 ml) was added to the compound (85.2 mg, 0.31 mmol)obtained in EXAMPLE 37 and refluxed in a Dean-Stark apparatus for 1 hr.p-toluene-sulfonic acid (catalytic amount) was added to the reactionmixture and refluxed again for 3 hrs. The solid, so formed, was filteredand purified by column chromatography with dichloromethane-methanol(15:1) to give the desired product (57.8 mg, 52%) as a pale brown solid.

IR(neat) 3400, 1743, 1658 cm⁻¹

¹H-NMR (400 MHz, CDCl₃:DMSO-d₆=3:1) δ 2.01-2.20 (m, 2H), 3.51-3.86 (m,2H), 5.315 (s, 2H), 5.34, 5.55 (ABq, 2H), 6.418 (s, 1H), 7.557 (s, 1H),7.676 (t, 1H), 7.83 (t, 1H), 8.03 (d, 1H), 8.18 (d, 1H), 8.570 (s, 1H)

MS(EI) m/e 364 (M⁺), 320 (M⁺−CO₂)

EXAMPLE 39

Preparation of dl-18-methoxymethyloxy camptothecin

Methoxymethyl chloride (6 μl, 0.08 mmol) and diisopropylethylamine (12μl, 0.07 mmol) was added to a suspension of dl-18-hydroxycamptothecin(20 mg, 0.05 mmol) in dichloromethane (2 ml) and stirred at 25 to 30° C.for 48 hrs.

With addition of dichloromethane (25 ml), the reaction mixture waswashed with a saturated ammonium chloride (10 ml), water (10 ml), 10%hydrogen sulfate potassium (10 ml), water (10 ml) and brine (10 ml)successively. The organic layer was dried over anhydrous magnesiumsulfate and concentrated under reduced pressure. The residue waspurified by column chromatography with dichloromethane-methanol (30:1)and PTLC with dichloromethane-methanol (20:1) to give the desiredproduct (5 mg, 23%) as a yellow solid.

IR (KBr) 3566, 1733, 1652 cm⁻¹

¹H-NMR (400 MHz, CDCl₃) δ 2.15-2.34 (m, 2H), 3.387 (s, 3H), 4.08-4.22(m, 2H), 4.604 (d, d, 2H), 5.313 (s, 2H), 5.62, 5.78 (ABq, 2H), 6.6 (s,1H), 7.68 (t, 1H), 7.726 (s, 1H), 7.84 (t, 1H), 7.96 (d, 1H), 8.24 (d,1H), 8.41 (s, 1H)

MS(EI) m/e 408 (M⁺) HRMS m/e M+Calcd; 408.1321, Obsd; 408.1336

In the same manner as in EXAMPLE 39, the compounds (R=H and n=2) ofEXAMPLE 40 to 46, expressed by the general formula (I), were prepared.

EXAM- PLE R₃ ¹H-NMR (CDCl₃ or CDCl₃ + CD₃OD) δ 40 OCOCH₃ 1.970(s, 3H),2.26-2.37(m, 2H), 4.126- 4.216(m, 2H), 5.32(s, 2H), 5.66, 5.79 (Aq, 2H),6.36(s, 1H), 7.65(s, 1H), 7.69(t, 1H), 7.86(t, 1H), 7.95 (d, 1H),8.25(d, 1H), 8.41(s, 1H) 41 OCOCH₂OCH₃ 8.3975(s, 1H), 8.2429(d, 1H,J=8.5Hz) 7.9401(d, 1H, J=8.3Hz), 7.8358(t, 1H), 7.6936(s, 1H), 7.6845(t,1H), 5.7928 5.3225(ABq, 2H, J=16.2Hz), 5.3064(s, 2H), 4,2762-4.2991(m,1H), 4.18-4.22 (m, 1H), 3.9794(d, 2H, J=2.4Hz), 3.4106(s, 3H),2.2333-2.2444(m, 2H) 42 OCH₂OCH₂CH₃ 8.4035(s, 1H), 8.2411(d, 1H,J=8.4Hz) 7.945(d, 1H, J=7.4Hz), 7.8409(t, 1H, J=7.0Hz), 7.7274(s, 1H),7.6744(t, 1H J=7.0Hz), 5.7673, 5.3189(ABq, 2H, J= 16.2Hz), 5.3132(s,2H), 4.6547(ABq, 2H), 4.3373(s, 1H), 4.2345(q, 2H), 3.81(m, 1H),3.7144(m, 1H), 2.22(m, 1H), 2.11(m, 1H), 1.2186(t, 3H) 43OCH₂OCH₂CH₂OCH₃ 8.4018(s, 1H), 8.2391(d, 1H, J=8.4Hz), 7.9423(d, 1H,J=7.6Hz), 7.8388(t, 1H), 7.7236(s, 1H), 7.6925(t, 1H), 5.7629,5.3223(ABq, 2H, J=16.3Hz), 5.3111(s, 2H), 4.6934(d, d, 2H), 3.8514(m,1H) 3.7011-3.7435(m, 2H), 3.65(m, 1H), 3.5602-3.5828(m, 2H), 3.3976(s,3H), 2.01-2.22(m, 2H) 44 OCONHPh 2.074-2.188(m, 1H), 2.308-2.361(m, 1H),4.122-4.163(m, 1H), 4.279-4.303(m, 1H) 5.153, 5.263(ABq, 2H, J=19.2Hz)5.464 (s, 2H), 6.707(t, 1H, J=7.2Hz) 7.013(t, 2H, J=8.0Hz), 7.202(d, 2H,J=8.0Hz), 7.350(s, 1H), 7.693(t, 1H J=7.2Hz), 7.842(t, 1H, J=8.4Hz),8.086(d, 1H, J=8.4Hz), 8.122(d, 1H, J=8.0Hz), 8.611(s, 1H), 9.337(brs,1H) 45 OCONHCH₂CH₂—Cl 9.19(brs, 1H), 8.3989(s, 1H), 8.2469 (d, 1H,J=8.4Hz), 7.9399(d, 1H, J=7.9 Hz), 7.8384(t, 1H), 7.7171(s, 1H),7.6727(t, 1H), 5.7667, 5.3430(ABq, 2H), 5.2748(q, 2H), 3.8914(m, 1H),3.78(m, 1H), 3.5472-3.5995(m, 2H), 3.4507-3.4833(m, 2H), 2.22(m, 2H) 46OCONH i-Pr. 8.3909(s, 1H), 8.2033(d, 1H, J=8.7Hz) 7.9206(d, 1H,J=7.5Hz), 7.8150(t, 1H) 7.7022(s, 1H), 7.6551(t 1H), 5.7112, 5.2815(ABq,2H, J=16.2Hz), 5.2778(s, 2H), 4.2720(m, 1H), 4.1706-4.1982(m, 1H),3.6538(m, 1H), 2.2597(m, 2H), 1.1270(d, 6H)

EXAMPLE 47

Preparation of6-cyano-1,1-(ethylenedioxy)-7-[1′-(ethoxycarbonyl)-2′-hydroxyethyl]-5-oxo-Δ⁶⁽⁸⁾-tetrahydroindolizine

A mixture of 35% formaldehyde (30 ml), dioxane (50 ml), water (20 ml)and ethanol (20 ml) was added to6-cyano-1,1-(ethylenedioxy)-7-[(ethoxycarbonyl)methyl]-5-oxo-Δ⁶⁽⁸⁾-tetrahydroindolizine(500 mg, 1.64 mmol) and stirred at 25 to 30° C. for 15 hrs. Withaddition of dichloromethane (120 ml), the reaction mixture was washedwith water(120 ml×3 times) and brine (120 ml). The separated organiclayer was dried over anhydrous magnesum sulfate and concentrated underreduced pressure. The residue was purified by column chromatography withdichloromethane-methanol (25:1) to give the desired product (318 mg,58%) as a white solid.

IR (KBr) 3310, 2224, 1735, 1647 cm⁻¹

¹H-NMR (400 MHz, CDCl₃) δ 1.275 (t, 3H, J=7.2 Hz), 2.407 (t, 2H, J=6.8Hz), 4.029-4.221 (,m, 9H), 4.239 (q, 2H), 6.383 (s, 1H)

MS(EI) m/e 334 (M⁺), 316 (M⁺−H₂O), 304 (M⁺−CH₃OH)

EXAMPLE 48

Preparation of6-cyano-1,1-(ethylenedioxy)-7-[1′-(ethoxycarbonyl)-2′-(methoxyethoxymethyloxyethyl)]-5-oxo-Δ⁶⁽⁸⁾-tetrahydroindolizine

Dichloromethane(0.7 ml) was added to the compound(51.6 mg, 0.15 mmol)obtained in EXAMPLE 47 and cooled in ice bath. Then, with a slowaddition of diisopropylethylamine (30 μl, 0.17 mmol) and MEM-Cl (35 μl,0.31 mmol), the resulting solution was stirred at 25 to 30° C. for 20hrs. To the reaction mixture, dichloromethane (15 ml) was added andwashed with a saturated sodium bicarbonate aqueous solution (10 ml×2times), water (10 ml) and brine (10 ml). The separated organic layer wasdried over anhydrous magnesium sulfate and concentrated under reducedpressure. The residue was purified by column chromatography withdichloromethane-methanol (25:1) to give the desired product (41 mg, 62%)as a white solid.

IR(neat) 2224, 1734, 1661 cm⁻¹

¹H-NMR (400 MHz, CDCl₃) δ 1.289 (t, 3H, J=6.8 Hz), 2.422 (t, 2H, J=6.8Hz), 3.410 (s, 3H), 3.548-3.598 (m, 2H), 3.653-3.680 (m, 1H),3.725-3.748 (m, 1H), 3.912 (dd, 1H, J=10 Hz, J=6.8 Hz), 4.098 (dd, 1H,J=10 Hz, J=6.8 Hz), 4.129-4.216 (m, 6H), 4.236 (q, 2H), 4.336 (t, 1H,J=6.8 Hz), 4.706, 4.742 (ABq, 2H, J=6.8 Hz), 6.441 (s, 1H)

MS(EI) m/e 423 (M⁺+H), 422 (M⁺), 392 (M⁺−OCH₃), 378 (M⁺−C₂H₅O)

EXAMPLE 49

Preparation of6-(acetamidomethyl)-1,1-(ethylenedioxy)-7-[1′-(ethoxycarbonyl)-2′-(methoxyethoxymethyloxyethyl)]-5-oxo-Δ⁶⁽⁸⁾-tetrahydroindolizine

With the compound(39.6 mg, 0.09 mmol) obtained in EXAMPLE 48, thereaction and work-up was carried out in the same procedure as in EXAMPLE33 and then, the residue was purified by column chromatography withdichloromethane-methanol (20:1) to give the desired compound(42 mg, 96%)as a colorless oil.

IR(neat) 1733, 1661, 1656 cm⁻¹

¹H-NMR (400 MHz, CDCl₃) δ 1.247 (t, 3H, J=7.2 Hz), 1.943 (s, 3H), 2.393(t, 2H, J=6.8 Hz), 3.405 (s, 3H), 3.546-3.569 (m, 2H), 3.660 -3.686 (m,2H), 3.809 (dd, 1H, J=6.4 Hz, J=9.2 Hz), 4.093-4.192 (m, 7H), 4.205 (g,2H), 4.467 (dq, 2H, J=6.4 Hz), 4.652 (t, 2H, J=6.8 Hz), 4.719, 4.750(ABq, 2H, J=6.8 Hz), 6.349 (s, 1H), 6.658 (s, 1H)

MS(EI) m/e 468 (M⁺), 453 (M⁺−CH₃), 425 (M⁺−C₂H₅O)

EXAMPLE 50

Preparation of6-(acetoxymethyl)-1,1-(ethylenedioxy)-7-[1′-(ethoxycarbonyl)-2′-(methoxyethoxymethyloxyethyl)]-5-oxo-Δ⁶⁽⁸⁾-tetrahydroindolizine

With the compound (359 mg, 0.76 mmol) obtained in EXAMPLE 49, thereaction and work-up was carried out in the same procedure as used inEXAMPLE 34 and then, the residue was purified by column chromatographywith dichloromethane methanol (30:1) to give the desired compound(234mg, 65%) as a colorless oil.

IR(neat) 1733, 1740, 1661 cm⁻¹

¹H-NMR (400 MHz, CDCl₃) δ 1.230 (t, 2H, J=7.2 Hz), 2.057 (s, 3H), 2.361(t, 2H, J=6.8 Hz), 3.381 (s, 3H), 3.521-3.542 (m, 2H), 3.620-3.653 (m,2H), 3.723 (dd, 1H, J=6.0 Hz, J=9.6 Hz), 4.082-4.209 (m, 8H), 4.225 (5,2H), 4.716-4.675 (ABq, 2H, J=6.8 Hz), 5.259 (s, 2H), 6.313 (s, 1H)

MS(EI) m/e 469 (M⁺), 426 (M⁺−COCH₃)

EXAMPLE 51

Preparation of6-(acetoxymethyl)-1,1-(ethylenedioxy)-7-[1′-(ethoxycarbonyl)vinyl]-5-oxo-Δ⁶⁽⁸⁾-tetrahydroindolizine

A mixture of dry benzene (8 ml) and DBU (416 μl, 2.78 mmol) was added tothe compound(522 mg, 1.1 mmol) obtained in EXAMPLE 50 and stirred atroom temperature for 3 hrs. With addition of dichloromethane (20 ml),the reaction mixture was washed with a saturated ammonium chlorideaqueous solution (15 ml×2) and water (15 ml). The separated organiclayer was dried over anhydrous magnesium sulfate and concentrated underreduced pressure. The residue was purified by column chromatography withdichloromethane-methanol (30:1) to give the desired product (363 mg,90%) as a yellowish white solid.

IR(neat) 1729, 1719, 1655 cm⁻¹

¹H-NMR (400 MHz, CDCl₃) δ 6.551 (s, 1H), 6.109, 5.773 (s, s, 1H×2),5.008 (s, 2H), 4.243 (q, 2H, J=7.2 Hz), 4.104-4.161 (m, 6H), 2.398 (t,2H, J=6.8 Hz), 2.021 (s, 3H), 1.293 (t, 3H, J=7.2 Hz)

MS(EI) m/e 363 (M⁺), 320

EXAMPLE 52

Preparation of 6-(acetoxymethyl)-1,1-(ethylenedioxy)-7-[1′-(ethoxycarbonyl)-1′-(hydroxy)-2′-(hydroxy)ethyl]-5-oxo-Δ⁶⁽⁸⁾-tetrahydroindolizine

Pyridine (3.9 ml) was added to the compound(285 mg, 1.79 mmol) obtainedin EXAMPLE 51, injected with OsO₄ (240 mg, 0.94 mmol)(0.08M toluene,11.8 ml) and stirred at room temperature for 4 hrs in the dark. Afterthe reaction was completed, the reaction mixture was added with asolution of NaHSO₃ (480 mg) and water (7 ml) and stirred for 1 hr. Thereaction mixture was extracted with dichloromethane (30 ml×5), and thecombined organic layers were washed with brine and dried over anhydrousmagnesium sulfate.

The organic layer was concentrated under reduced pressure. The residuewas purified by column chromatography with dichloromethane-methanol(20:1) to give the desired product (280 mg, 90%) as a white solid.

IR(neat) 3307, 1735, 1654 cm⁻¹

¹H-NMR (400 MHz, CDCl₃) δ 6.455 (s, 1H), 5.303 (ABq, 2H, J=11. 7 Hz),4.611 (s, 1H) 4.085-4.611 (m, 9H), 3.855, 3.826 (dd, 1H, J=6.8, 11.2Hz), 2.641 (brt, 1H, J=6.8 Hz), 2.384 (t, 2H, J=6.8 Hz), 2.063 (s, 3H),1.290 (t, 3H, J=6.8 Hz)

MS(EI) m/e 397 (M⁺), 354

EXAMPLE 53

Preparation of 1,1-(ethylenedioxy)-(5′-hydroxymethyl-5′-hydroxy-2′H,5′H,6′H-6-oxopyrano)[3′,4′-f]-5-oxo-Δ⁶⁽⁸⁾-tetrahydroindolizine

With the compound(325 mg, 0.82 mmol) obtained in EXAMPLE 52, thereaction and work-up was carried out in the same procedure as in EXAMPLE35 and then, the residue was purified by column chromatography withdichloromethane-methanol (20:1) to give the desired compound (211 mg,84%) as a white solid.

IR(neat) 3421, 1750, 1654 cm⁻¹

¹H-NMR (400 MHz, CDCl₃) δ 6.599 (s, 1H), 5.564, 5.255 (ABq, 2H, J=16.0Hz), 4.072-4.238 (m, 7H), 3.772, 3.733 (dd, 2H, J=11.6 Hz), 2.418 (t,2H, J=6.8 Hz)

MS(EI) m/e 309 (M⁺), 293, 280, 279, 265

HRMS m/e (M⁺) Calcd: 309, 0849, Obsd: 309, 0.847

EXAMPLE 54

Preparation of1,1-(ethylenedioxy)-(5′-acetoxymethyl-5′-hydroxy-2′H,5′H,6′H-6-oxopyrano)[3′,4′-f]-5-oxo-Δ⁶⁽⁸⁾-tetrahydroindolizine

The compound (119 mg, 0.38 mmol) obtained in EXAMPLE 53 was dissolved inanhydrous dichloromethane (10 ml) and with addition of pyridine (93 μl,1.15 mmol) and acetic anhydride (47 μl, 0.50 mmol), the reaction mixturewas stirred at room temperature for 20 hrs. The reaction mixture wasdiluted with dichloromethane (20 ml), washed with 10% potassiumbisulfate aqueous solution (20 ml), water (20 ml) and brine (20 ml), anddried over anhydrous magnesium sulfate.

The organic layer was concentrated under reduced pressure. The residuewas purified by column chromatography with dichloromethane-methane(20:1) to give the desired compound (127 mg, 94%) as a white solid.

IR(neat) 3437, 1751, 1657 cm⁻¹

¹H-NMR (400 MHz, CDCl₃) δ 6.586 (s, 1H), 5.636, 5.295 (ABq, 2H, J=16.4Hz), 4.302 (d, 1H, J=11.2 Hz), 4.116-4.215 (m, 7H), 4.004 (s, 1H), 2.427(t, 2H, J=6.8 Hz), 2.084 (s, 3H)

In the same procedure as used in EXAMPLE 54, the compounds of EXAMPLE 55to 57 were prepared and the results were as follows:

EXAMPLE R₃ ¹H-NMR(CDCl₃)δ 55 OCONHPh 8.205(brs, 1H), 7.260-7.436(m, 4H),7.055(d, 1H), 6.661(s, 1H), 5.587, 5.357(ABq, 2H), 4.366, 4.319(d, d,2H, J=11.2Hz), 4.101-4.227(m, 6H), 2.413(t, 2H, J=6.8Hz) 56 OCONH i-Pr.6.61(s, 1H), 5.64, 5.56(ABq, 2H), 4.66(d, 1H), 4.53(d, 1H), 4.1253(m,6H), 3.7981-3.8826(m, 1H), 2.4203(t, 2H), 1.1478(d, 6H) 57

6.5831(s, 1H), 5.6356, 5.3385(ABq, 2H, J=16.4Hz), 4.3821(d, 1H, J=11.4Hz), 4.2693(d, 1H, J=11.4Hz), 4.1275-4.2176(m, 6H), 4.0542(d, 2H,J=2.0Hz), 3.4240(s, 3H), 2.4235(t, 2H, J=6.8Hz)

EXAMPLE 58

Preparation of1,5-dioxo-(5′-acetoxymethyl-5′-hydroxy-2′H,5′H,6′H-6-oxopyrano)[3′,4′-f]-Δ⁶⁽⁸⁾-tetrahydroindolizine

80% trifluoroacetic acid(1.3 ml) was added to the compound (127 mg, 0.36mmol) obtained in EXAMPLE 54 and stirred at room temperature for 2 hrs.The reaction mixture was concentrated under reduced pressure and withaddition of brine, the reaction mixture was extracted withdichloromethane (20 ml×3) and dried over anhydrous magnesium sulfate.The organic layer was concentrated under reduced pressure. The residuewas purified by column chromatography with dichloromethane-methanol(20:1) to give desired product (theoretical yield) as a yellow solid.

IR (KBr) 3412, 1746, 1660 cm⁻¹

¹H-NMR (400 MHz, CDCl₃) δ 7.318 (s, 1H), 5.687, 5.388 (ABq, 2H, J=17.1Hz), 4.405 (t, 2H), 4.370 (d, 1H, J=11.7 Hz), 4.155 (d, 1H, J=11.7 Hz),3.007 (t, 2H, J=6.8 Hz), 2.080 (s, 3H)

MS(EI) m/e 307 (M⁺)

In the same manner as used in EXAMPLE 58, the compounds (n=1) of EXAMPLE59 to 61, expressed by the general formula (III),were prepared from thecompounds of Examples 55, 56, and 57, respectively.

EXAMPLE R₃ ¹H-NMRδ 59 OCONHPh 8.646(brs, 1H), 7.339(s, 1H), 7.156-7.356(m, 5H), 6.092(s, 1H), 5.573, 5.31(ABq, 2H), 4.359, 4.318(d, d, 2H)4.267(t, 2H), 2.885(t, 2H) 60 OCONH i-Pr. 7.2509(s, 1H), 5.65,5.3550(ABq, 2H, J=17.2Hz), 4.71(d, 1H), 4.52(d, 1H), 4.3554(t, 2H,J=6.8Hz), 3.7965-3.8806 (m, 1H), 2.9616(t, 2H, J=6.8Hz), 1.1335.(d, 6H)61

used immediately to the next reaction without further purification

EXAMPLE 62

Preparation of dl-20-desethyl-20-acetoxymethyl camptothecin

With the compound(19 mg, 0.06 mmol) obtained from EXAMPLE 58 andN-(2-aminobenzylidene)-p-toluidine (20 mg, 0.09 mmol), the reaction andwork-up was carried out in the same procedure as in EXAMPLE 38 and then,the residue was purified by PTLC with dichloromethane-methanol (20:1) togive the desired compound(9 mg, 36%) as a brown solid.

IR(CHCl₃) 3400, 1749, 1740, 1654 cm⁻¹

¹H-NMR (400 MHz, CDCl₃+CD₃OD=4:1) 8.493 (s, 1H), 8.223 (d, 1H, J=8.4Hz), 7.988 (d, 1H, J=8.0 Hz), 7.867 (t, 1H, J=7.2 Hz), 7.767 (s, 1H),7.703 (t, 1H, J=7.2 Hz), 5.697, 5.433 (ABq, 2H, J=16.4 Hz), 5.334 (s,2H), 4.460 (d, 1H, J=11.2 Hz), 4.330 (d, 1H, J=11.2 Hz), 2.087 (s, 3H)

LRMS(EI) m/e 392 (M⁺), 331, 320, 305

HRMS m/e (M⁺) Calcd: 392.1008, Obsd: 392.1006

In the same manner as in EXAMPLE 38, the compounds (R=H and n=1) ofEXAMPLE 63 to 65, expressed by the general formula (I), were prepared.

EXAMPLE R₃ ¹H-NMR(CDCl₃ or CDCl₃+CD₃OD)δ 63 OCONHPh 8.501(s, 1H),8.228(d, 1H, J=8.0Hz), 8.000(d, 1H, J=8.0Hz), 7.879(t, 1H, J=7.8Hz),7.381(s, 1H), 7.714(t, 1H, J=7.8Hz), 7.361(d, 2H, J=8.0Hz), 7.244(t, 2H,J=7.2Hz), 7.017(t, 1H, J=7.2Hz), 5.705, 5.561(ABq, 2H, J=16.8Hz),5.328(s, 2H), 4.518(d, 1H, J=11.2Hz), 4.395(d, 1H, J=11.2Hz) 64 OCONHi-Pr 9.8022(d, 1H), 8.4071(s, 1H), 8.2465 (d, 1H, J=8.4Hz), 7.9457(d,1H, J=8.0Hz) 7.8480(t, 1H, J=7.0Hz), 7.7197(s, 1H), 7.6783(t, 1H,J=7.0Hz), 5.7705, 5.4392 (ABq, 2H, J=16.4Hz), 5.4598(s, 2H), 4.4212(d,1H, J=11.8Hz), 4.2169(d, 1H, J=11.8Hz), 3.7709-3.8068(m, 1H) 1.2605(d,6H) 65

8.4077(s, 1H), 8.2476(d, 1H, J=8.7Hz) 7.9451(d, 1H, J=7.1Hz), 7.8447(t,1H) 7.6998(s, 1H), 7.6787(t, 1H), 5.7736, 5.4860(ABq, 2H, J=16.6Hz),5.3195(s, 2H), 4.5546(d, 1H, J=11.4Hz), 4.3254 (d, 1H, J=11.4Hz),4.0714(d, 2H, J=1.0Hz), 3.4257(s, 3H)

EXAMPLE 66

Preparation of dl-20-desethyl-20-hydroxymethylcamptothecin

A mixture of methanol (6 ml), water (2 ml) and LiOH.H₂O (33 mg, 0.78mmol) was added to dl-20-desethyl-20-acetoxymethylcamptothecin (140 mg,0.36 mmol) and the reaction mixture was stirred at room temperature for1.5 hrs.

The reaction mixture was concentrated under reduced pressure to removemethanol and stirred in an ice bath for 30 mins until the pH of thesolution became controlled 3-3.5 with 1N HCl. The solid, so formed, wasfiltrated, washed with water, isopropanol and ether successively, andunder P₂O₅, dried in vacuum for 3 hrs to give the desired product(83 mg,66%) as a yellowish white soild.

IR(KBr) 3436, 1743, 1657 cm⁻¹

1H-NMR (400 MHz, CDCl₃+DMSO-d₆=1:1) δ 8.608 (s, 1H, 8.157 (d, 1H, J=8.0Hz), 8.106 (s, 1H), 8.050 (d, 1H, J=8.0 Hz), 7.829 (t, 1H, J=7.3 Hz),7.673 (t, 1H, J=7.3 Hz), 6.741 (s, 1H), 5.441, 5.360 (ABq, 2H, J=16.1Hz), 5.304 (s, 2H), 3.850-3.89 (m, 1H), 3.681-3.723 (m, 1H)

LRMS(EI) m/e 350 (M⁺), 320, 306

HRMS m/e (M⁺) Calcd: 350.0903, Obsd: 350. 0917

In the same manner as in EXAMPLE 39, the compounds (R=H and n=1) ofEXAMPLE 67 to 69, expressed by the general formula (I), were prepared.

EXAM PLE R₃ ¹H-NMR (CDCl₃) δ 67 OCH₂OCH₃ 8.397(s, 1H), 8.240(d, 1H,J=8.0Hz), 7.939(d, 1H, J=80Hz), 7.838(t, 1H, J=8.0Hz), 7.700(s, 1H),7.670(t, 1H, J=8.0Hz), 5.713, 5.410(ABq, 2H, J=16.4Hz), 5.313(s, 2H),4.626, 4.587(ABq, 2H, J=6.8Hz), 4.187(s, 1H), 3.839(d, d, 2H, J=10.4Hz),3.315(s, 3H) 68 OCH₂OEt 8.4192(s, 1H), 8.2621(d, 1H, J=8.4Hz) 7.9602(d,1H, J=8.4Hz), 7.8588(t, 1H) 7.7240(s, 1H), 7.6917(t, 1H, J=8.0Hz)5.7287, 5.4221(ABq, 2H, J=16.3Hz), 5.3344(s, 2H), 4.7030, 4.6508(ABq,2H, J=6.9Hz), 4.2620(s, 1H), 3.8727(q 2H, J=7.0Hz), 3.6212-3.6628(m,1H), 3.5079-3.5554(m, 1H), 1.2136(t, 3H, J=7.0Hz) 69 OCH₂OCH₂CH₂—OCH₃8.3955(s, 1H), 8.1848(d, 1H, J=8.4Hz) 7.9156(d, 1H, J=7.6Hz), 7.8082(t,1H) 7.7868(s, 1H), 7.6908(t, 1H), 5.6227, 5.3642(ABq, 2H, J=16.4Hz),5.2757(s, 2H), 4.6619(d, 1H, J=6.9Hz), 4.6218 (d, 1H, J=6.9Hz),3.8422(dd, 2H, J=2, 3, 10.0Hz), 3.6112-3.6380(m, 1H), 3.5719-3.5952(m,1H), 3.4664-3.4924(m, 2H), 3.3192(s, 3H)

Experiment 1: Cytotoxicity of the Compounds According to this Invention

1) Materials

Cancer Cell Lines

In the determination of cytotoxicity, the following cell lines,purchased from ATCC (U.S.A.), were used for the experiments as thetarget organisms: L1210 (ATCC CCL 219), A172 (ATCC CRL 1620), A427 (ATCCHTB 53), A549 (ATCC CCL185), SK-NEP-1 (ATCC HTB 48), CAOV-3 (ATCC HTB75), HEC-1-B (ATCC HTB 113), DLD-1 (ATCC CCL 221) and KATO-III (ATCC HTB103), CAKI-2 (ATCC HTB 47).

Preparation of Culture Medium for Cancer Cells

Distilled water was used for the preparation of culture medium. 1 literof powdered RPMI 1640 medium was dissolved in distilled water and withaddition of NaHCO₃ (2.0 g), the resulting solution was stirred. Afterthe pH of the solution was adjusted to 6.8 to 7.4, the medium wasfiltered by 0.22 μm filter. The fetal bovine serum was thermally treatedat 56° C. for 30 mins before use.

Reagents

RPMI 1640 culture medium for cancer cells, fetal bovine serum, sodiumbicarbonate and trypsin-EDTA buffer were purchased from Gibco Co. AndMTT (3,4,5-dimethylthiazol -2-yl) -2,5-diphenyltetrazolium bromide) andsulforhodamine B(SRB) reagent were obtained from Sigma Co. and othergeneral reagents were of G.R. degree.

Test materials Camptothecin, purchased from Sigma Co., was dissolved indimethylsulfoxide and with addition of Dulbec co's phosphate bufferedsaline (DPBS), the concentration of dimethylsulfoxide was adjusted to10% before use. In case of the synthesized test materials dissolved indimethylsulfoxide, the concentration of dimethylsulfoxide was alsoadjusted to 10%, while the other packed with DPBS. The finalconcentration of dimethyl sulfoxide given to cancer cells was adjustedto less than 1%, which had no influence on the growth of cancer cells.

2) Methods

The cancer cells were grown at 37° C., 5% CO₂ in RPMI 1640 mediumcontaining 10% (v/v) fetal serum (FBS), 50 μg/ml gentamicin and 2 g/Lsodium bicarbonate. (In order to detach anchorage-dependent cell linesgrown by adhering to a culture flask) the medium was removed, washedwith PBS one time and with addition of 2 to 3 ml of trypsin-EDTA buffer,the monolayers of cancer cells were wholly covered to stand for the timebeing. Then, a suspension of the cultured cancer cells was prepared,diluted with a medium and inoculated on 96-well plate so that the numberof cells may be the same as described in the following Table 1.

20 μl of sample (PBS solution in a control well) was inoculated to96-well microplate and cultured for 3 days. After cultivation, theanti-neoplastic activites were measured by SRB assay [Test Scheme 1].

As for anchorage-independent cell such as L1210, cells were inoculatedso that the number of cell may also be the same as described in thefollowing Table 1. After being cultured for 2 days, the cytotoxicityagainst the cells were measured by MTT assay [Test Scheme 2].

The optical density was measured using an automatic microplate reader.The concentration of cell-growth inhibition rate by 50% was calculatedby GI₅₀ (computer PCS version 4.1 by Probit method). As per theexperiment one time, the mean of optical density on about 2 to 3 wellswas used for the calculation.

TABLE 1 Number of inoculated cells per No. of inoculated Cell LineOrigin cells (well) Property (Human) A427 or A549 Lung 5,000 monolayerA172 CNS 5,000 DLD-1 Colon 5,000 HEC-1-b Endometrium 5,000 CAOV-3 Ovary20,000 SK-NEP-1 Kidney 20,000 or CAKi-2 KATO-III Stomach 10,000 (Mice)L1210 Leukemia 14,000 suspension

TABLE 1 Number of inoculated cells per No. of inoculated Cell LineOrigin cells (well) Property (Human) A427 or A549 Lung 5,000 monolayerA172 CNS 5,000 DLD-1 Colon 5,000 HEC-1-b Endometrium 5,000 CAOV-3 Ovary20,000 SK-NEP-1 Kidney 20,000 or CAKi-2 KATO-III Stomach 10,000 (Mice)L1210 Leukemia 14,000 suspension

Test Scheme 2: MTT assay 180 μl of cell suspension in medium + 20 μl ofsample or PBS ↓ Cultured at 37° C. for 2 days, 5% carbon dioxide. ↓Added with 50 μl of MTT solution (2 mg/ml in PBS solution). ↓ Culturedfor 4 hrs. ↓ Centrifuged at 2,000 rpm for 10 mins. Removal ofsupernatant. ↓ Dissolved formazan crystals in DMSO. ↓ Measurement ofoptical density at 570 nm. ↓ Calculation of GI₅₀

3) Results

The cytotoxicity results of each cell line were as table 2 and 3:

TABLE 2 Example NO. A427 A172 DLD-1 HEC-1-B CAOV-3 SK-NEP-1 KATO IIIL1210 MEAN 18 0.14 0.73 0.65 1.8 1.21 0.0077 8.6 0.43 1.83 19 0.31 1.320.31 4.7 0.08 0.013 0.29 0.62 1 26 0.23 0.15 0.17 1.37 0.09 0.0031 0.462.18 0.35 29 0.07 0.05 0.26 7.68 0.12 0.015 0.21 0.38 1.19 30 0.0570.065 0.064 1.65 0.025 0.00077 0.024 0.08 0.27 38 0.83 1.63 0.91 20.52.3 0.17 3.54 2.3 4.27 39 0.45 1.07 0.76 1.34 0.23 0.018 0.13 0.18 0.5740 2.4 1.62 2.86 2.57 1.72 0.12 0.4 2.07 1.67 44 2.6 0.028 0.58 10.3 1.83.1 2.6 3.32 3 Camptothecin 0.032 0.017 0.047 0.081 0.02 0.00019 0.0920.024 0.041

TABLE 3 Example NO. A549 A172 DLD-1 HEC-1-B CAOV-3 SK-NEP-1 KATO IIIL1210 MEAN 20 0.08 0.54 1.04 4.22 0.023 0.061 1.05 0.73 0.968 21 2.5111.34 9.68 10.16 2.74 25.16 10.17 5.55 9.66 25 0.94 1.09 5.53 11.44 0.657.55 7.86 1.65 4.59 27 3.43 0.4 1.87 7.54 0.47 0.17 0.44 2.34 2.08 280.98 11.54 4.35 12.33 2.29 8.89 23.64 6.45 8.81 41 1.15 3.05 13.88 16.232.79 1.90 1.33 10.71 6.38 42 0.092 0.58 0.45 1.99 0.78 0.82 8.08 0.61.674 43 0.25 1.83 6.84 8.88 0.87 0.99 23.98 2.25 5.74 46 0.56 2.36 4.9225.27 0.64 3.11 20.88 4.23 7.75 62 0.05 0.6 0.48 5.15 0.32 2.45 3.7 0.671.68 63 0.34 1.3 1.99 7.95 0.74 6.17 6.13 1.3 3.24 64 3.28 4.89 10.239.75 9.79 51.3 31.48 2.6 19.16 65 2.06 7.43 19.91 20.2 5.45 7.66 53.695.75 15.27 66 0.88 2.52 6.24 6.43 0.76 10.79 4.04 1.39 4.13 67 0.2 3.310.95 5.4 1.58 2.78 9.17 0.65 3.01 68 1.72 4.55 6.98 14.23 7.18 16.316.64 1.39 8.62 69 1.55 1.92 11.32 19.78 2.63 2.13 36.61 9.73 10.71Camptothecin 0.02 0.05 0.07 0.4 0.01 0.2 0.4 0.06 0.2

Experiment 2: Antitumor activities of the Compounds According to thisInvention

The antitumor activities may be usually proven by determining tumorgrowth inhibitory effects on solid tumor and prolongation effects oflife span on artificially tumor-bearing animals.

Antitumor activities were measured through the experiments onprolongation effects of life span against L1210-bearing mice and tumorgrowth inhibitory effects against B16-bearing mice. To evaluate theantitumor activities on L1210 leukemia, a certain number of L1210 cells(in vitro) was intraperitoneally inoculated for propagation and theL1210 cells in ascites were harvested from animals sacrificed bycervical dislocation on 7th day after tumor was implanted.

To investigate tumor growth inhibitory effects against B16 melanoma, acertain number of B16 cells(in vitro) was subcutaneously inoculated forpropagation and tumor cells were resected from animals scrificed bycervical dislocation on 14th day after tumor was implanted.

The following is a detailed description of this EXPERIMENT for antitumoractivities.

Experimental Animals

Male BDF₁ mice, weighing about 20 grams, were used. Animal rooms werecontrolled at 23±2° C. and 60±5% relative humidity, under HEPA filter,and maintained in specific pathogen free (SPF).

Experiment 2-1: Prolongation Effects of Life Span against L1210-bearingMice in Accordance with the Compounds of this Invention.

5×10⁵ of L1210 leukemia cell, obtained in vitro form, wereintraperitoneally inoculated in BDF₁ mice for propagation. The L1210tumor cells in ascites were aseptically harvested from BDF₁ micesacrificed by cervical dislocation on 7th day after tumor implantation.

The tumor cells were passaged at least three times prior to use in thetest.

5×10⁵ cell were intraperitoneally inoculated into BDF₁ mice on day 0 andthe sample (0.1 ml/log) were intraperitoneally administration on days 1,3 and 5. six mice were used for each experimental group.

Antitumor activities were evaluated by the mean survival time of a groupof mice and also expressed by the ILS(%) value and the mean animalweights for day 1 and day 5.

Increased Life span (ILS)

ILS(%)={(MST of sample treated group/MST of solvent group)−1}×100

Results

The compound of example 18 given 1st, 3rd and 5th days after the day oftumor inocualtion increased the life span (ILS) of L1210-bearing mice by33% and 64% at total doses of 10 mg/kg and 50 mg/kg, respectively (Tabel4).

TABLE 4 Prolongation effects of life aginst L1210 in the compound ofexample 18 Dose Survival days 30 days B.W. Compound (mg/kg) MST ILS(%)survivor Change(g) Control 9.75 0 0/6 0 Compound of 10 13.00 33 0/6−0.25 example 18 Compound of 50 16.00 64 0/6 −0.80 example 18

The compound of example 26 given 1st, 3rd and 5th days after the day oftumor inoculation increased the life span (ILS) of L1210-bearing mice by72% and 75% at total doses of 10 mg/kg and 50 mg/kg, respectively (Table5).

TABLE 5 Prolongation effects of life against L1210 of the compoundprepared in example 26 Dose Survival days 30 days B.W. Compound (mg/kg)MST ILS(%) survivor Change(g) Control 9.125 0 0/6 +1.77 Compound of 1015.67 72 0/6 −0.09 example 26 Compound of 50 16.00 75 0/6 −0.57 example26

The compound of example 30 given 1st, 3rd and 5th days after the day oftumor inoculation increased the life span (ILS) of L1210-bearing mice by42% and 119% at total doses of 10 mg/kg and 50 mg/kg, respectively(Table 6).

TABLE 6 Prolongation effects of life against L1210 of the compoundprepared in example 30 Dose Survival days 30 days B.W. Compound (mg/kg)MST ILS(%) survivor Change(g) Control 9.125 0 0/6 +1.77 Compound of 1013.00 42 0/6 −0.71 example 30 Compound of 50 20.00 119 0/6 −0.57 example30

Since the compound of example 30 exhibited better dose dependency andILS(%) than other compound, the inventor et al. tried to elucidate ahigher dose level and amounts showing antitumor activities inexperimental animals administered with a higher dose.

When the compound of example 30 was given 1st, 3rd and 5th days afterthe inoculation at total doses of 8.75, 17.5, 35, 70 and 140 mg/kg, theILS(%) showed 58.7, 79.7, 92.4, 143 and 110%, respectively (Table 7).

TABLE 7 Prolongation effects of life against L1210 of the compoundprepared in example 30 in various dose Dose Survival days 30 days B.W.Compound (mg/kg) MST ILS(%) survivor Change(g) Control 9.875 0 0/6 +1.44Compound of 8.75 15.67 58.7 0/6 +0.7 example 30 Compound of 17.5 17.7529.2 0/6 +0.45 example 30 Compound of 35 19.00 92.4 0/6 −0.12 example 30Compound of 70 24.00 143 0/6 −0.79 example 30 Compound of 140 20.75 1100/6 −0.93 example 30

When the compound of example 30 was given on day 1, 3 and 5 after thetumor inoculation, the maximum ILS of 143% was obtained at total dose of70 mg/kg.

The antitumor activity of the compound of example 30 againstintraperitoneally-implanted L1210 was evaluated in parallel with that ofcamptothecin as shown in FIG. 1. The compound of example 30 gave higherILS(%) and wider range where the effect was siginificant.

Experiment 2-2: Tumor Growth Inhibitory Effects on B16-bearing Mice inAccordance with the Compounds of this Invention

10⁶ of B16 melanoma cell, obtained in vitro form, were subcutaneously inBDF₁ mice for propagation. The B16 melanomas were transfered on day 14.The fresh tumors except for necrotized center were aseptically resectedfrom tumor-bearing mice sacrificed by cervical dislocation.

The tumor cells were passaged at least injected three times prior to usein the test.

1 g subcutaneously donor tumor to be subcutaneously was homogenized incold sterilized saline to give a final concentration of 1 g/10 ml. The1:10 tumor suspension is injected into all BDF₁ mice subcutaneouslyusing 0.2 ml/mouse.

The mice were subcutaneously implanted with such homogenized solutionand 7 to 8th day after the tumor implantation, the mice having a certainsize of tumors were selected for experiments. Each experimental groupwere administered with the sample (0.1 ml/10 g) on 8th, 12th and 16thday after the inoculation of tumor. Eight mice were used for eachexperimental group.

The size of the tumors was calculated by direct measurement of thediameter of the tumors, using the formula.

Tumor size=(a ² b)/2

Where b is the largest diameter and a is the diameter perpendicular to band growth curves were thus obtained. (FIG. 2)

The inhibition rate of tumor growth on the basis of tumor size wascalculated according to the formula.

Wherein;

IR(%)=(1−TW_(t)/TW_(c))×100

TW_(t) is the mean tumor volume(weight) of sample-treated groups.

TW_(c) is the mean tumor volume(weight) of control group.

Test Results

When the compound of example 30 was given 8, 12 and 16 days after thetumor inoculation at total doses of 35, 70 and 140 mg/kg , the IR(%)showed 27, 54.8 and 67.72%, respectively (Table 8).

TABLE 8 Tumor growth inhibitory effects against B16 melanoma of thecompound prepared in example 30 2. Compound of 3. Compound 4. Compoundof 1. Camptothecin example 30 of example 30 example 30 Control 10 mg/kg35 mg/kg 70 mg/kg 140 mg/kg Tumor size day 8  87.69 ± 13.58  85.95 ±13.15  86.93 ± 11.34  86.21 ± 10.12 86.94 ± 9.34 day 12 598.50 ± 93.59458.56 ± 53.87 411.88 ± 71.52 320.06 ± 71.79 328.98 ± 76.57 day 162492.75 ± 437.80 1035.98 ± 129.36 1234.65 ± 182.64 870.46 ± 77.27 695.75± 82.77 day 20 4194.83 ± 285.66 1775.50 ± 300.91 3061.34 ± 752.341897.88 ± 256.22 1353.91 ± 134.36 IR (%) 57.8% 27% 54.8% 67.72% Tumorweight day 20  3.92 ± 0.32  1.61 ± 0.30  2.34 ± 0.44  1.71 ± 0.18  1.11± 0.21 IR (%) 59% 40% 56% 71.6%

Futher, 20th day after the compound of example 30 was administered, thepercentage of IR on the weight of the incised tumor showed 40, 56 and71.6%, respectively.

The maximum IR of 67.72% was obtained at total dose of 140 mg/kg. Thechanges in tumor volumes were outlined in FIG. 2. Such results clearlyimply that the compound of example 30 has not only better antitumoractivity on ascites tumor and solid type tumor, but also less toxicityand a wide range of safety margin, compared with camptothecin.

What is claimed is:
 1. A camptothecin compound of the formula I, or apharmaceutically acceptable salt thereof

wherein: R is hydrogen or −(CH₂)₂—NR₁R₂, where R₁ is hydrogen or anamino protecting group and R₂ is a lower alkyl, hydroxyethyl oracetoxyethyl group, or R₁ and R₂, together with the nitrogen atom towhich they are attached, form a cyclic moiety; n is 1 or 2; and R₃ ishydrogen or −OR₄, where R₄ is hydrogen, COR₅, CONHR₆, or CH₂OR₇, whereR₅ is methyl or CH₃OCH₂—, R₆ is isopropyl, phenyl or —CH₂CH₂Cl and R₇ ismethyl, ethyl, or CH₃OCH₂CH₂—; with the provisos that: R and R₃ cannotboth be hydrogen; when n is 2 and R is —(CH₂)₂—N(Cbz)CH₃, then R₃ cannotbe hydrogen; when n is 2 and R₃ is OH, then R cannot be hydrogen; andwhen R is —CH₂CH₂NHCH₃, then R₃ cannot be hydrogen.
 2. An antineoplasticcomposition containing a camptothecin compound of the formula I, or apharmaceutically acceptable salt thereof, and a pharmaceuticallyacceptable carrier

wherein: R is hydrogen or —(CH₂)₂—NR₁R₂, where R₁ is hydrogen or anamino protecting group and R₂ is a lower alkyl, hydroxyethyl oracetoxyethyl group, or R₁ and R₂, together with the nitrogen atom towhich they are attached, form a cyclic moiety; n is 1 or 2; and R₃ ishydrogen or —OR₄, where R₄ is hydrogen, COR₅, CONHR₆, or CH₂OR₇, whereR₅ is methyl or CH₃OCH₂—, R₆ is isopropyl, phenyl or —CH₂CH₂Cl and R₇ ismethyl, ethyl, or CH₃OCH₂CH₂—; with the provisos that: R and R₃ cannotboth be hydrogen; when n is 2 and R is —(CH₂)₂—N(Cbz)CH₃, then R₃ cannotbe hydrogen; when n is 2 and R₃ is OH, then R cannot be hydrogen; andwhen R is —CH₂CH₂NHCH₃, then R₃ cannot be hydrogen.
 3. A process formanufacturing a compound of the formula I by reacting a compound of theformula II with a compound of the formula III under acidic conditions

wherein: R′ is hydrogen or −(CH₂)₂—NR₁R₂, where R₁ is an aminoprotecting group and R₂ is a lower alkyl, hydroxyethyl or acetoxyethylgroup, or R₁ and R₂, together with the nitrogen atom to which they areattached, form a cyclic moiety; P is hydrogen or —(CH₂)₂—NR₁R₂, where R₁is hydrogen or an amino protecting group and R₂ is a lower alkyl,hydroxyethyl or acetoxyethyl group, or R₁ and R₂, together with thenitrogen atom to which they are attached, form a cyclic moiety; n is 1or 2; and R₃ is hydrogen or —OR₄, where R₄ is hydrogen, COR₅, CONHR₆, orCH₂OR₇, where R₅ is methyl or CH₃OCH₂—, R₆ is isopropyl, phenyl or—CH₂CH₂Cl and R₇ is methyl, ethyl, or CH₃OCH₂CH₂—; with the provisosthat: R and R₃ cannot both be hydrogen; when n is 2 and R is—(CH₂)₂—N(Cbz)CH₃, then R₃ cannot be hydrogen; when n is 2 and R₃ is OH,then R cannot be hydrogen; and when R is —CH₂CH₂NHCH₃, then R₃ cannot behydrogen.